Sequential HNCACB and CBCANH protein NMR pulse sequences.

The pulse sequences HNCACB and CBCANH correlating side chain C(beta) resonances with amide resonances in the protein backbone do not distinguish between inter- and intraresidue correlations. The new pulse sequences sequential HNCACB and sequential CBCANH make this distinction by suppressing coherence transfer between 13C(alpha) and 15N via the one-bond J(NC(alpha)) coupling so that only the sequential correlations are observed in the spectrum. The experimental results of applying sequential HNCACB in a clean-TROSY-adapted implementation to the protein Chymotrypsin Inhibitor 2 at 800 MHz are presented.

Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance.

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.

A sequential HNCA NMR pulse sequence for protein backbone assignment.

The conventional HNCA pulse sequence suffers from the ambiguity that it cannot distinguish inter- and intraresidue correlations because the one-bond and two-bond J(NC(alpha)) coupling constants are of similar magnitude. This paper presents a novel pulse sequence, sequential HNCA, that leads to a spectrum exhibiting exclusively interresidue correlations. This important sequential information has so far usually been obtained by an HN(CO)CA experiment that for medium field strengths typically also is more sensitive than HNCA. However, for increasing static magnetic fields the chemical shift anisotropy relaxation mechanism of carbonyl carbons becomes more and more efficient, leading to a degradation of the HN(CO)CA sensitivity. Hence there is a point where the sequential HNCA experiment becomes the most sensitive option for sequential N-C(alpha) correlation.

Editing and diagonal peak suppression in three-dimensional HCCH protein NMR correlation experiments.

A novel three-dimensional (3D) HCCH NMR experiment is introduced. It involves 13C-13C COSY or TOCSY coherence transfer plus two independent editing steps according to the number of protons attached to the individual carbons before and after the 13C-13C homonuclear mixing. This double editing leads to simplification of HCCH protein side chain spectra that otherwise are prone to spectral overlap. Another interesting feature is amino acid selectivity, i.e. that the presence of certain correlations in a doubly edited HCCH subspectrum gives a clue as to assignment to a particular subgroup of amino acids or segments thereof. Finally, the selection of two different multiplicities in the two editing steps leads to diagonal peak suppression in the 1H-1H (3D spectrum recorded with two 1H and one 13C dimension) or the 13C-13C (3D spectrum recorded with one 1H and two 13C dimensions) two-dimensional projection. The new experiment is demonstrated using a 13C,15N-labeled protein sample, chymotrypsin inhibitor 2, at 500 MHz.

Clean TROSY: compensation for relaxation-induced artifacts.

TROSY pulse sequences for recording, e.g., (1)H-(15)N chemical shift correlation spectra of proteins are designed to select only one of four two-dimensional multiplet components. However, all of the variants published so far are prone to relaxation-induced artifacts at the positions of two of the other multiplet components. This article introduces modifications to the two spin-state-selective coherence transfer building blocks of the TROSY mixing sequence resulting in a clean TROSY spectrum with the artifacts largely suppressed. It works by having the new mixing sequence generate peaks of opposite phase at the positions of the relaxation artifacts. The clean TROSY pulse sequence is marginally shorter than the original one and contains the same pulses. Experimental demonstration is presented for the (15)N-labeled proteins RAP 17-97 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein) and EQT, equinatoxin II, from the Mediterranean anemone Actinia equina.

Suppression of diagonal peaks in three-dimensional protein NMR TROSY-type HCCH correlation experiments.

A novel method for suppression of (13)C-(13)C diagonal peaks without sensitivity loss in three-dimensional HCCH TROSY-type NMR correlation experiments involving aromatic side chains in proteins (Pervushin et al., J. Am. Chem. Soc. 120, 6394-6400 (1998)) is presented. The key element is a spin-state-selective filter in the (13)C-(13)C mixing sequence with the dual effect of selecting the TROSY resonance in the preceding evolution period and interchanging TROSY and anti-TROSY resonances. The cross peaks are invariant to this filter but diagonal peak coherence gets concentrated on the anti-TROSY transition so that it can be eliminated by a (13)C --> (1)H TROSY transfer element. The new method is demonstrated using a (13)C,(15)N-labeled protein sample, RAP 18-112 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein), at 750 MHz.

New techniques for the measurement of C'N and C'H(N) J coupling constants across hydrogen bonds in proteins.

Two new two- or three-dimensional NMR methods for measuring (3h)J(C'N) and (2h)J(C'H) coupling constants across hydrogen bonds in proteins are presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D or 3D spectra into two separate subspectra corresponding to the two possible spin states of the (1)H(N) spin during evolution of (13)CO coherences. This allows (2h)J(C'H) to be measured in an E.COSY-type way while (3h)J(C'N) can be measured in the so-called quantitative way provided a reference spectrum is also recorded. A demonstration of the new methods is shown for the (15)N,(13)C-labeled protein chymotrypsin inhibitor 2.

3hJ coupling between C(alpha) and H(N) across hydrogen bonds in proteins.

J couplings between (13)C(alpha) and (1)H(N) across hydrogen bonds in proteins are reported for the first time, and a two- or three-dimensional NMR technique for their measurement is presented. The technique exploits the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms, for sensitivity enhancement. The 2D or 3D spectra exhibit E.COSY patterns where the splittings in the (13)CO and (1)H(N) dimensions are (1)J((13)C(alpha), (13)CO) and the desired (3h)J((13)C(alpha), (1)H(N)), respectively. A demonstration of the new method is shown for the (15)N,(13)C-labeled protein chymotrypsin inhibitor 2 where 17 (3h)J((13)C(alpha), (1)H(N)) coupling constants ranging from 0 to 1.4 Hz where identified and all of positive sign.

The structure of the membrane-binding 38 C-terminal residues from bovine PP3 determined by liquid- and solid-state NMR spectroscopy.

The secondary structure and membrane-associated conformation of a synthetic peptide corresponding to the putative membrane-binding C-terminal 38 residues of the bovine milk component PP3 was determined using 1H NMR in methanol, CD in methanol and SDS micelles, and 15N solid-state NMR in planar phospholipid bilayers. The solution NMR and CD spectra reveal that the PP3 peptide in methanol and SDS predominantly adopts an alpha-helical conformation extending over its entire length with a potential bend around residue 19. 15N solid-state NMR of two PP3 peptides 15N-labelled at the Gly7 and Ala32 positions, respectively, and dissolved in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol phospholipid bilayers shows that the peptide is associated to the membrane surface with the amphipathic helix axis oriented parallel to the bilayer surface.

Suppression of diagonal peaks in TROSY-type 1H NMR NOESY spectra of 15N-labeled proteins.

A novel method for suppression of diagonal peaks in the amide region of NOESY NMR spectra of 15N-labeled proteins is presented. The method is particularly useful for larger proteins at high magnetic fields where interference between dipolar and chemical shift anisotropy relaxation mechanisms results in large TROSY effects, i.e. , large differences in 1HN linewidths depending on the spin state of attached 15N nuclei. In this limit the new TROSY NOESY method does not compromise sensitivity. It is demonstrated using a perdeuterated 15N-labeled protein sample, Neural Cell Adhesion Molecule 213-308 (NCAM) from rat, in H2O at 800 MHz.

Pulse sequences for measurement of one-bond (15)N-(1)H coupling constants in the protein backbone.

A set of three improved two-dimensional (2D) NMR methods for measuring one-bond (15)N-(1)H coupling constants in the protein backbone is presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D spectra into two separate subspectra corresponding to the two possible spin states of the coupling partner. Cross talk between the two subspectra is a second order effect in the difference between the actual coupling constants and the one used in setting the pertinent delays of the pulse sequences. This relatively high degree of editing accuracy makes the methods useful for applications to molecules subjected to weak alignment where the one-bond coupling constants are linear combinations of a scalar J and a residual dipolar contribution containing important structural information. A demonstration of the new methods is shown for the (15)N-labeled protein chymotrypsin inhibitor 2 in a lipid bicelle mixture.

The role of coherence transfer efficiency in design of TROSY-type multidimensional NMR experiments.

An improved method for TROSY-type (Pervushin et al., Proc. Natl. Acad. Sci. USA 94, 12366-12371 (1997)) heteronuclear two-dimensional correlation involving protons of negligible CSA is presented. Rather than applying a simple INEPT sequence for back-transfer to protons (Pervushin et al., J. Am. Chem. Soc. 120, 6394-6400 (1998)), we replace the pi/2 proton pulse in INEPT by a spin-state-selective coherence transfer element (Sorensen et al., J. Biomol. NMR 10, 181-186 (1997)) and maintain broadband decoupling during acquisition. Theoretically that results in a sensitivity enhancement of a factor of 2. The new method is demonstrated using a (13)C,(15)N-labeled protein sample, RAP 18-112 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein), at 750 MHz.

Spin-state-selective TPPI: a new method for suppression of heteronuclear coupling constants in multidimensional NMR experiments.

A novel multidimensional NMR pulse sequence tool, spin-state-selective time-proportional phase incrementation (S(3) TPPI), is introduced. It amounts to application of different TPPIs on the two components of doublets so that their frequencies can be manipulated independently. The chief application is for suppression of large heteronuclear one-bond coupling constants in indirect dimensions of multidimensional experiments without interchanging the two transverse magnetization components of doublets as conventional decoupling does, which is advantageous when they relax at different rates such as by partial compensation of dipolar and CSA relaxation contributions. For experimental confirmation we use a sample of (15)N-labeled neural cell adhesion molecule modules 1 and 2, a protein with a molecular weight of about 20 kDa. The new tool is general and can be combined with many multidimensional NMR experiments for proteins.

Optimization of three-dimensional TROSY-type HCCH NMR correlation of aromatic (1)H-(13)C groups in proteins.

Improved methods for three-dimensional TROSY-Type HCCH correlation involving protons of negligible CSA are presented. The TROSY approach differs from the conventional approach of heteronuclear decoupling in evolution and detection periods by not mixing fast and slowly relaxing coherences and usually suppressing the former. Pervushin et al. (J. Am. Chem. Soc. 120, 6394-6400 (1998)) have proposed a 3D TROSY-type HCCH experiment where the TROSY approach is applied only in one of the (13)C dimensions. A new pulse sequence applying the TROSY approach in both indirect dimensions is advantageous when the TROSY effect of the carbons is large or when a relatively high resolution is required. For lower resolutions or moderate TROSY effects we show that it is possible to combine the best of both worlds, namely to suppress heteronuclear couplings without mixing fast and slowly relaxing coherences while at the same time superimpose the two components and thus have both contribute to the detected signal. That is possible using the novel technique of Spin-State-Selective Time-Proportional Phase Incrementation (S(3) TPPI). The new 3D S(3) TPPI TROSY HCCH method is demonstrated on a (13)C,(15)N-labeled protein sample, RAP 18-112 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein), at 750 MHz and average sensitivity enhancements of 10% are obtained for the cross peaks in comparison to methods based on conventional decoupling on one of the carbons or on TROSY on both carbons.

13C natural abundance S3E and S3CT experiments for measurement of J coupling constants between 13Calpha or 1Halpha and other protons in a protein.

It is demonstrated that the spin-state-selective pulse sequence elements, S3E and S3CT, previously introduced for measurement of J coupling constants in 15N-labeled proteins can be applied for work with peptides and proteins with 13C at the natural abundance level. In addition, a method is described for suppression of crosstalk caused by passive spin flips and pulse imperfections, which otherwise results in systematically underestimated J coupling constants and thereby inaccurate structural constraints. This method is also applicable for crosstalk suppression in applications of S3E and S3CT to 13C- or 15N-labeled samples. Experimental confirmation is obtained using a 10 mM BPTI sample focusing on 13C in the alpha position. The measured J coupling constants include 3J(HN-Halpha) and 3J(Halpha-Hbeta) related to the phi and chi1 angles, respectively.

Simultaneous and independent rotations with arbitrary flip angles and phases for I, ISalpha, and ISbeta spin systems.

A new pulse sequence element for simultaneous and independent rotations with arbitrary flip angles and phases for isolated I, ISalpha, and ISbeta resonances without the use of selective radiofrequency pulses is introduced and experimentally demonstrated. S is a directly attached heteronucleus either at natural abundance or isotopically enriched. This pulse sequence element, dubbed TIG-BIRD (triselective independent gyrations BIRD), generalizes earlier elements like BIRD, TANGO, BANGO, and BIG-BIRD, the latter of which allows for arbitrary selection of flip angles and phases for I and IS spin systems without discriminating between ISalpha and ISbeta resonances. For ISalpha and ISbeta spin systems it also generalizes the spin-state-selective excitation (S3E) element selectively exciting only one of the ISalpha or ISbeta resonances. TIG-BIRD is a nonselective addition to the NMR toolkit which effects the equivalent of three independent selective rotations for I, ISalpha, and ISbeta resonances.

New methylene specific experiments for the measurement of scalar spin-spin coupling constants between protons attached to 13C.

New two- and three-dimensional NMR methods are proposed for the measurement of 3J(H, H) coupling constants between two adjacent methylene moieties. The new experiment, which is based on a combination of the E.COSY principle and double/zero quantum heteronuclear spectroscopy, has been applied to diaceton-glucose and to the protein rhodniin. The coupling constants of CH-CH2 groups have been compared with those obtained from a HCCH-E.COSY experiment to check the reliability of the results. An analysis of the coupling constants derived by comparison between experimental and simulated spectra is presented. Simulations were done with the program wtest considering fully correlated dipolar relaxation. Side-chain conformations in amino acids with adjacent methylene groups can be determined by the new experiment.

New multidimensional editing experiments for measurement of amide deuterium isotope effects on Cbeta chemical shifts in 13C, 15N-labeled proteins.

Novel multidimensional NMR pulse sequences for measurement of the three- and four-bond amide deuterium isotope effect on the chemical shifts of 13Cbeta in proteins are presented. The sequences result in editing into two subspectra of a heteronuclear triple resonance spectrum ¿omega(N), omega(Cbeta), omega(Halpha)¿ according to there being a deuterium or a proton attached to 15N for the pertinent correlations. The new experiments are demonstrated by an application to the first module of the 13C,15N-labeled protein RAP 18-112 (N-terminal module of alpha2-macroglobulin receptor associated protein).